Impact of Amino Acid Substitutions near the Catalytic Site on the Spectral Properties of an O2-Tolerant Membrane-Bound [NiFe] Hydrogenase
| Autor: | Oliver Lenz, Bärbel Friedrich, Miguel Saggu, Friedhelm Lendzian, Robert Bittl, Peter Hildebrandt, Ingo Zebger, Marcus Ludwig |
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| Rok vydání: | 2010 |
| Předmět: |
Hydrogenase
Stereochemistry Cupriavidus necator Inorganic chemistry Enzyme catalysis Residue (chemistry) Mutant protein Catalytic Domain Spectroscopy Fourier Transform Infrared Physical and Theoretical Chemistry chemistry.chemical_classification biology Electron Spin Resonance Spectroscopy Active site biology.organism_classification Atomic and Molecular Physics and Optics Protein Structure Tertiary Amino acid Oxygen Amino Acid Substitution chemistry Mutagenesis Site-Directed biology.protein Cysteine |
| Zdroj: | ChemPhysChem. 11:1215-1224 |
| ISSN: | 1439-7641 1439-4235 |
| DOI: | 10.1002/cphc.200900988 |
| Popis: | [NiFe] hydrogenases are widespread among microorganisms and catalyze the reversible cleavage of molecular hydrogen. However, only a few bacteria, such as Ralstonia eutropha H16 (Re), synthesize [NiFe] hydrogenases that perform H(2) cycling in the presence of O(2). These enzymes are of special interest for biotechnological applications. To gain further insight into the mechanism(s) responsible for the remarkable O(2) tolerance, we employ FTIR and EPR spectroscopy to study mutant variants of the membrane-bound hydrogenase (MBH) of Re-carrying substitutions of a particular cysteine residue in the vicinity of the [NiFe] active site that is characteristic of O(2)-tolerant membrane-bound [NiFe] hydrogenases. We demonstrate that these MBH variants, despite minor changes in the electronic structure and in the interaction behavior with the embedding protein matrix, display all relevant catalytic and noncatalytic states of the wild-type enzyme, as long as they are still located in the cytoplasmic membrane. Notably, in the oxidized Ni(r)-B state and the fully reduced forms, the CO stretching frequency increases with increasing polarity of the respective amino acid residue at the specific position of the cysteine residue. We purified the MBH mutant protein with a cysteine-to-alanine exchange to apparent homogeneity as dimeric enzyme after detergent solubilization from the membrane. This purified version displays increased oxygen sensitivity, which is reflected by detection of the oxygen-inhibited Ni(u)-A state, an irreversible inactive redox state, and the light-induced Ni(a)-L state even at room temperature. |
| Databáze: | OpenAIRE |
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